Mucin is an important glycoprotein of the mucous that covers the trachea, the digestive tract such as the stomach, lumina such as the gonads, and the like. Mucin has countless sugar chains that are bonded to polypeptides (core proteins) through O-glycoside bonds. The core proteins of mucin are coded by various mucin genes (MUC genes). Important roles of mucin are to protect and hydrate as well as to lubricate mucous membranes. Mucin also participates in regulating the differentiation and regeneration of the epithelium, cell adhesion, the immune response, cell signaling, and the like. In recent years, numerous genes coding for many core proteins of mucin have been cloned, and their full or partial sequences have been determined. Most mucins have many repetitive sequence domains (tandem repeats). These tandem repeats are comprised of amino acid sequences (tandem units) of varying length, and are rich in serine, threonine, and proline residues. Many O-linked sugar chains of various structures are added to these serine or threonine residues. In the completed mucin, residues that are not considered to be 0-glycosylated (naked peptides) are present in a constant ratio. Generally, the sugar chains are comprised of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), galactose (Gal), fucose (Fuc), sialic acid (SA), mannose (Man), and the like. Mucin comes in the forms of secreted mucin that is produced by the epithelial cells and the like, and membrane-bonded mucin that has a hydrophobic transmembrane site and is present in a state bonded to cell membranes.
The core proteins of mucin are collectively referred to as mucin, with numbers being assigned in order of discovery. In humans, 19 genes coding for these core proteins have been reported (MUC1, 2, 3A, 3B, 4, 5AC, 5B, 6, 7, 8, 9, 11, 12, 13, 15, 16, 17, 18, 19). Of these, 11 are transmembrane mucin and seven are secreted mucin (Nonpatent Reference 1).
MUC4 is present in the epithelial tissue of the trachea, colon, stomach, ectocervix, lungs, and the like (Nonpatent Reference 1). MUC4 is known to be associated with various diseases, such as cancer.
The extracellular domains of human MUC4 contain various numbers of tandem units of 16 amino acid residues having seven potential O-glycosylation sites. In O-glycan, the sugars that are initially transferred to the serine and threonine residues of the core protein through O-bonds in greatest quantity are GalNAc, followed by Man, Fuc, GlcNAc, and the like. O-glycans are incompletely processed by cancer cells, and cause the expression of common sugar antigens Tn (GalNAcα-1-Ser/Thr), STn (NeuAcα2-6 GalNAcα1-O-Ser/Thr), and T (Galβ1-3 GalNAcα-1-O-Ser/Thr) with cancer. After the initial sugar, sugars are transferred one after another and the O-glycan sugar chain grows longer. Many core structures of the O-glycan to which GalNAc is initially transferred are known and have been numbered. The main currently known core structures are indicated below. Longer sugar chains of more complex structure grow based on these structures.
Core 0 (Tn antigen) GalNAc
Core 1 (T antigen) Galβ1-3GalNAc
Core 2: Galβ1-3(GlcNAcβ1-6) GalNAc
Core 3: GlcNAcβ1-3GalNAc
Core 4: GlcNAcβ1-3(GlcNAcβ1-6) GalNAc
Core 5: GalNAcα1-3GalNAc
Core 6: GlcNAcβ1-6GalNAc
Core 7: GalNAcα1-6GalNAc
Core 8: Galα1-3GalNAc
Core 9: Galβ1-3(Galβ1-6) GalNAc
Core 10: GalNAcβ1-3GalNAc
Core 11: GalNAcβ1-3(GalNAcβ1-6) GalNAc
Core 12: Galβ1-3(Glcβ1-6) GalNAc
Core 13: Galβ1-3(Glcβ1-4) (Glcβ1-6) GalNAc
The addition of sugar chains by the O-glycosylation of mucin core proteins plays important roles in the protection of the outer layer of epithelial cells, immune reactions, cell adhesion, inflammatory reactions, carcinogenesis, and cancerous metastasis. Among the mucins, much research has been conducted on MUC1 mucin. The overexpression of MUC1 in carcinogenesis and the connection between the dramatic change in O-glycosylation and carcinogenesis and metastasis have been reported. Further, research and development of diagnostic and therapeutic drugs for lung cancer and ovarian cancer employing monoclonal antibodies to MUC1 is advancing (Nonpatent Reference 2, Patent References 1 and 2).
Marked fluctuation in the expression of core proteins accompanying carcinogenesis has been reported for MUC4 (Nonpatent Reference 1). Overexpression of MUC4 in pancreatic and esophageal cancer has been found to accelerate cancer proliferation and metastasis (Nonpatent Reference 1). Additionally, by inhibiting expression of the MUC4 gene in cancer cells, the proliferation of cancer cells is markedly inhibited, and it has become clear in in vivo investigation that cell migration, cell adhesion, and aggregation are accelerated. It has been indicated that were it possible to inhibit the function of MUC4, it would be possible to impede the progress and metastasis of metastatic cancer (Nonpatent Reference 3). Further, the fact that interaction between MUC4 and galectin is important in the metastatis of pancreatic cancer and the like has been recently discovered (Nonpatent References 4 and 5).    Patent Reference 1: WO2010/050528    Patent Reference 2: WO2011/135869    Patent Reference 3: JP-A-2006-111618    Patent Reference 4: WO2011/054359    Nonpatent Reference 1: Chaturvedi et al., FASEB J. 22: 966-981 (2008)    Nonpatent Reference 2: Beatson et al., Immunotherapy 2: 305-327 (2010)    Nonpatent Reference 3: Singh et al., Cancer Res. 64: 622-630 (2004)    Nonpatent Reference 4: Liu and Rabinovich, Nature Rev. Cancer 5: 29-41 (2005)    Nonpatent Reference 5: Sanapati et al., Clin Cancer Res 17:267-274 (2011)    Nonpatent Reference 6: Moniaux et al., J. Histochem. Cytochem. 52: 253-261(2004)    Nonpatent Reference 7: Zhang et al., J. Cellular Physiol. 204: 166-177 (2005)    Nonpatent Reference 8: Matsushita et al., Biochemistry 52:402-414 (2013)    Nonpatent Reference 9: Hashimoto et al., Chem. Eur. J. 17: 2393-2404 (2011)    Nonpatent Reference 10: Ohyabu et al., J. Am. Chem. Soc. 131: 17102-17109 (2009)    Nonpatent Reference 11: Matsusita et al., Biochim. Biophy. Acta 1840: 1105-1116 (2014)    Nonpatent Reference 12: Sanapati et al., Clin Cancer Res 17:267-274 (2011)
A number of monoclonal antibodies to purified MUC4 of unspecified structure and recombinant and synthetic peptides of the MUC4 gene have been manufactured in the past. For example, a monoclonal antibody to the MUC4β, unit peptide in the form of IG8 (Nonpatent Reference 7) and a monoclonal antibody to the MUC4 tandem unit peptide STGDTTPLPVTDTSSV in the form of 8G7 (Nonpatent Reference 6) have been achieved and are employed as research tools. There are reports of monoclonal antibodies to glycopeptides derived from MUC4 in the form of 4D9, 3C9, 6E3, and 6C11 (Patent Reference 4). However, they are all antibodies with low specificity in which the sugar modification sites and numbers are not specified for the MUC4 peptide.
Accordingly, the present invention has for its object to provide an antibody specific to MUC4 having a sugar chain structure that is highly expressed in cancer cells, a glycopeptide serving as an antigen suited to the preparation of this antibody, and a new means and method of diagnosing, preventing, and/or treating cancer based on this antibody.
The present inventors demonstrated by NMR that structurally specific conformational change was induced in the main chain peptides of the sugar chains bonded to side chains in the epitope region of the antibody, that peptide conformation was sensitively changed by sugar chain modification in specific amino acid residues, and that this specified the antigen structure in the MUC1 antibody (Nonpatent Reference 8). Analysis of change in the three-dimensional structure of synthetic glycopeptides derived from mucin by MS and NMR has revealed that the conformation of glycopeptides was affected by sugar chain modification of the multiple threonine residues present in the peptide, and the new knowledge that sugar chain modification at specific sites imparted stable conformation of the peptide main chain (Nonpatent Reference 9). Additionally, the present inventors synthesized many O-linked sugar amino acids and glycopeptides, including the compounds described in (Patent Reference 3), to elucidate the various structures and functions of the sugar chains.
Further, a highly sensitive, high-performance immobilized glycopeptide microarray that is capable of accurate antibody specificity analysis and epitope mapping has been developed, and a new method of determining the true epitope structure has been established (Nonpatent References 10 and 11).